Exploiting HUH-endonucleases for bioconjugation of mammalian proteins.

Abstract

The rep domain of HUH-endonucleases process single-stranded DNA (ssDNA) in nature during rolling circle replication, conjugative plasmid transfer, and DNA integration into the host genome by bacteria and viruses. Upon the recognition of a highly conserved nonanucleotide located in a hairpin, HUH-endonucleases catalyze the cleavage reaction of ssDNA, forming a covalent protein-DNA intermediate which has been exploited as a fusion tag for bioconjugation in several biotechnology applications. “HUH-tags” are appealing for their ability to covalently link proteins to DNA in a specific manner and in a ‘one-pot’ reaction which is an inexpensive alternative to other methods that often require the modification of DNA bases or additional steps. To expand the application of HUH-tags for use in mammalian cells and in fusion with mammalian proteins, a panel of HUH-endonucleases from several viral families were characterized for their secretion and catalytic activity in HEK293 cells. Based on robust expression and activity,the WDV rep was used to attach DNA handles to the Notch1 mechanosensing domain for single molecule studies using optical tweezers. Additionally, WDV was expressed with a number of ligands involved in mechanosensing proteins on the surface of cells and the ECM to attach ssDNA for the development of Tension Gauge Tether DNA-based molecular tension sensors.